Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acylgalactosylceramide (AGC) synthesis was measured in vivo, and in a cell free system. 24 hours post-injection of [3H] palmitic acid into rat brain, more than 60% of the AGC radioactivity was associated with an ester linkage. Isolated rat myelin was incubated in the presence of [14C] palmitic acid, 2mM ATP, 50 microM CoA and 10 mM MgCl2 and acylation of myelin cerebrosides occurred at a linear rate for at least 60 min. Incubation of isolated myelin under standard conditions with [3H] cerebrosides and [14C] palmitic acid produced double labeled AGC. Labeling of AGC was maximum at pH 7.5 and 37 degrees C and appeared to be enzyme mediated inasmuch as it was reduced by myelin incubation with trypsin and drastically reduced by preheating the myelin for 5 min at 80 degrees C. Omission of ATP, CoA, MgCl2 or all three did not reduce fatty acid incorporation into AGC when compared to the values in the complete system. Addition of Triton X100 or Sodium Dodecyl Sulfate had little or no effect on the acylation of cerebrosides. Pulse chase experiments indicated that the reaction involved the net addition of fatty acid to the cerebrosides, rather than a rapid fatty acid exchange.
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PMID:Intramyelinic conversion of cerebrosides into acylgalactosylceramides. 262 89

1. Two forms of fatty acid-binding proteins (FABPs) were isolated from human, pig and rat liver cytosols by gelfiltration and anion-exchange chromatography. 2. Both forms did not show physicochemical or chemical differences. They had an Mr of about 14.5 kDa for all species. pI Values were 5.8 for both forms of human and pig liver FABP and 6.4 for both forms of rat liver FABP. In contrast to heart FABPs no tryptophan was present in liver FABPs. 3. Liver FABPs show a much higher enhancement of fluorescence at binding of 11-dansylaminoundecanoic acid, 16-anthroyloxy-palmitic acid and 1-pyrene-dodecanoic acid than heart FABPs and additionally a blue shift in excitation and emission wavelengths with the first fatty acid. 4. The bulky side-chain did not affect fatty acid binding since binding constants of liver FABPs were comparable for these fluorescent fatty acids and oleic acid (0.3-0.7 microM). 5. A 1:1 binding stoichiometry was obtained for oleic acid binding with heart and liver FABPs. 6. Liver FABPs have a high binding affinity for C16-C22 saturated and unsaturated fatty acids, palmitoyl-CoA, bromo-substituted fatty acids, POCA, tetradecylglycidic acid and flavaspidic acid. 7. Fatty acid binding could be reduced to less than 50% by arginine modification with 2,3-butadione or by enzymatic degradation of FABPs with trypsin or pronase.
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PMID:The binding affinity of fatty acid-binding proteins from human, pig and rat liver for different fluorescent fatty acids and other ligands. 274 9

Affinity labeling with palmitic acid was used to identify long chain fatty acid-binding sites of bovine serum albumin. [1-14C]Palmitic acid was activated by esterification with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K). The product was purified by chromatography and shown to compete with unesterified fatty acids for binding sites on bovine serum albumin. Activated [14C]palmitic acid coupled covalently to albumin producing [14C]palmitoyl-albumins containing from 0.12 to a maximum of 6.9 mol of attached label per mol of albumin. The presence of the covalently attached affinity label depressed binding of other long chain fatty acids to albumin. Albumin carrying 1 eq. of [14C]palmitate was cleaved using cyanogen bromide, pepsin, and trypsin. Radioactive peptides were isolated by high pressure liquid chromatography. Three peptides accounted for greater than 90% of the label. Residues labeled with [14C]palmitate were identified as Lys-116, Lys-349 and Lys-473, and the relative distribution of label was 10, 45, and 45% respectively, consistent with the presence of two strong binding sites in the COOH-terminal half of albumin and a somewhat weaker site in the NH2-terminal half.
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PMID:Location of long chain fatty acid-binding sites of bovine serum albumin by affinity labeling. 309 94

A preparation of peptidyl-tRNA from intact microsomes of mucin-synthesizing polysomes of sublingual salivary gland cells contained fatty-acylated galactosamine-free and galactosamine-enriched peptidyl-tRNA fractions, whereas trypsin-chymotrypsin treated microsomes yielded predominantly the acylated galactosamine-enriched peptidyl-tRNA complexes. Radioscanning and chemical analyses revealed that palmitate was substituted on all nascent peptides, except those shorter than 20 amino-acid residues. In contrast, the [35S]-methionine label was detected only on galactosamine-free peptides containing up to 70 amino acids. On SDS-polyacrylamide gel, the peptides released from galactosamine-enriched tRNA complexes separated into a multitude of bands ranging in size from 6000 to 60,000 dalton, whereas the total preparation afforded peptides ranging from 2000 to 60,000 dalton. Pulse-chase experiments, using radiolabelled methionine, palmitic acid and N-acetylgalactosamine, combined with chemical characterization of the radiolabelled fatty acids and carbohydrates from purified peptidyl-tRNA, confirmed that the N-terminal fatty acylation and the initial O-glycosylation with N-acetylgalactosamine are the co-translational processes taking place as soon as peptide is sufficiently large to be acylated, trimmed, and translocated to the luminal site of endoplasmic membrane.
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PMID:Co-translational processing and intracellular transport of rat salivary mucus glycoprotein. 325 86

The aim of this study was to analyze whether a monoclonal antibody to human milk fat globule membrane-associated antigens, recognized specifically and homogeneously by human breast carcinoma cells but also by normal epithelial cells active in secretion, could be used to restrict the access of antitumoral drugs to cells exposing the epitope. The drug-antibody conjugate to be used is constructed by means of a covalent peptidic linkage stable in extracellular medium but hydrolyzed by lysomal enzymes after endocytosis of the drug-carrier conjugate. This monoclonal antibody specifically immunoprecipitates radioactive material from MCF-7 cells biosynthetically radiolabeled with galactose, glucosamine, palmitic acid, or acetic acid but not with mannose, leucine, or methionine. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol, the label migrates as two bands with apparent molecular weights of about 350,000 and 400,000. These bands disappear, or their molecular weight is affected, after treatment of the cells with cycloheximide or of cell lysates with trypsin, Pronase, or neuraminidase but not treatment of the immunoprecipitate with endoglycosidase F. This suggests that these antigens are glycoproteins with O-linked oligosaccharides containing sialic acid in the epitope. By analogy, they should be similar, if not identical, to those recognized by the monoclonal antibodies designated HMFG1 (H. Burchell, H. Durbin, and J. Taylor-Papadimitriou, J. Immunol., 131:508-513, 1983) and DF3 (H. Sekine, T. Ohno, and D.W. Kufe, J. Immunol., 135:3610-3615, 1985). Binding at 4 degrees C of the 3H-labeled antibody by MCF-7 cells indicates the specific attachment of about 1.2 X 10(6) IgG molecules per cells with a Kd of about 14 nM. At 37 degrees C, cells take up the 3H-labeled antibody in amounts much higher than the binding capacity. In addition to cell-associated material, labeled digestion products are released into the culture medium. Cell fractionation by differential centrifugation and isopycnic equilibration on sucrose gradient indicates that the bulk of cell-associated antibody is distributed like the marker enzyme of lysosomes. Although the total uptake of the antibody by the cells is unaffected by either 50 microM chloroquine or 3 micrograms/ml cycloheximide, the release of digestion products is completely inhibited by chloroquine. Antigen-antibody dissociation is pH dependent, since, respectively, 50 and 84% of membrane-bound antibody are released during washing at pH 4.6 and 4.1.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Binding and endocytosis of a monoclonal antibody to a high molecular weight human milk fat globule membrane-associated antigen by cultured MCF-7 breast carcinoma cells. 336 2

Rubella virus contains three major structural polypeptides designated E1, E2, and C with molecular weights of 62,000, 47,000-54,000 (a complex), and 38,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced conditions. Limited-digest peptide maps confirm that each of these polypeptides is distinct and the E2 is a series of three closely related glycopolypeptides. Both E1 and E2 are glycosylated and covalently incorporate [3H]palmitic acid. Enzymatic digestion of intact virus with trypsin completely degrades both E1 and E2, while the C polypeptide remains intact. E1 has an isoelectric point of pH 6.5. E2 exhibits at least 15 different isoelectric species, which focus over the pH range of 5.0-8.6, and C has two distinct isoelectric species of pH 8.8 and pH 9.5. Under unreduced conditions, E1 exists as a disulfide-bonded dimer (E1-E1) with a molecular weight of 105,000; a disulfide-bounded heterodimer (E1-E2) with a molecular weight of 95,000; and in monomeric form (E1). E2 is found predominantly in heterodimeric form (E1-E2), and C is found only in dimeric form when unreduced. Functional-inhibition studies with selected monoclonal antibodies show at least three distinct antigenic domains on E1 that include sites involved in hemagglutination and lysis of red blood cells.
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PMID:A model of the structural organization of rubella virions. 400 20

We have studied the synthesis and expression of surface proteins in zygotes of Plasmodium gallinaceum during their transformation to mature ookinetes. The cells were biosynthetically labelled in vitro using [35S]methionine and proteins were immunoprecipitated with rabbit anti-ookinete serum or monoclonal antibodies. Early zygotes (approx. 2 h post-gametogenesis and fertilization) synthesized and expressed on their surface a protein of Mr 26 000 as observed under reducing conditions on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (31 000 under non-reducing conditions) and continued to do so for 8-10 h; thereafter synthesis of the Mr 26 000 protein declined and little or none was synthesized in the mature ookinetes (greater than 20 h post-gametogenesis). Between 3-5 h post-gametogenesis, zygotes also began to synthesize a protein of Mr 28 000 (34 000 under non-reducing conditions). Synthesis and expression of this surface protein continued throughout development; and the Mr 28 000 protein was the predominant surface protein synthesized by the mature ookinete. Mr 26 000 and Mr 28 000 proteins have been designated earlier as PgO-1 and PgO-2 respectively (Carter and Kaushal, Mol. Biochem. Parasitol. (1984) 13, 235-241). Neither protein was synthesized in the gametocytes prior to gametogenesis. Both proteins could be labelled with [3H]glucosamine or [3H]mannose. When zygotes were incubated with [3H]palmitic acid both PgO-1 and PgO-2 bound fatty acids in covalent linkage. The two proteins do not otherwise appear to be structurally related. They were differentially immunoprecipitated by different monoclonal antibodies and gave rise to distinct patterns of peptides following digestion with proteases such as Staphylococcus aureus V-8, trypsin and chymotrypsin.
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PMID:Biosynthesis of two stage-specific membrane proteins during transformation of Plasmodium gallinaceum zygotes into ookinetes. 403 6

Infection of BHK 21 cells by vesicular stomatitis virus (VSV) results in the intracellular synthesis of the five viral proteins which are easily detectable in polyacrylamide gels after short labeling periods with [35S]methionine. In addition, a 6th prominent radioactive protein band appears intracellularly in VSV-infected BHK cells. This additional polypeptide is also coded by the viral genome, because it is immunoprecipitated by antibodies against viral particles and more specifically by antibodies against purified G-protein. We propose to call this derivative of the G-protein Gsi-protein (short intracellular G-protein). It is associated with intracellular membranes and has an apparent mol. wt. of 58 000. Both G- and Gsi-protein have the same kinetics of appearance in the cell. The ratio of G-:Gsi-protein in BHK 21 cells is approximately 85:15. The mol. wt. difference of approximately 6000 daltons between G- and Gsi-protein is not due to variations in the degree of glycosylation because trypsin digestions of both [3H]mannose-labeled glycoproteins gave rise to identical glycopeptide patterns. Incubation of microsomes with trypsin demonstrates that Gsi-protein is protected in its full length by intracellular membranes. Gsi-protein is lacking an extended carboxy-terminal region of the viral G-protein sequence because it is not modified by palmitic acid and is not immunprecipitated by specific antibodies against a C-terminal peptide of the G-protein. Limited proteolysis by endoproteinase arg C indicates that the structure of Gsi-protein is very similar to the shedded form of the G-protein which has been previously described in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intracellular appearance of a glycoprotein in VSV-infected BHK cells lacking the membrane-anchoring oligopeptide of the viral G-protein. 608 25

Previously a new small subclass of SV40 large T antigen with a high-binding affinity to living target cells was characterized (J. Lange-Mutschler and R. Henning, 1983, Virology 127, 333-344.) In the present study the external binding process, particularly the tight linkage of T antigen to lipid of the target cells, was analyzed. Extraction of SV40-transformed target cells (SV80) first by sonification yielded approx 80% of [35S]methionine-labeled T antigen (mechanical extract). A further 20% was obtained by treatment of cellular debris with hydroxylamine (hydroxylamine extract). As shown by an 125I-protein A radioimmunoassay, hydroxylamine extracts contained significantly higher amounts of cell surface binding T antigen. Correspondingly, after incubating [3H]palmitic acid-prelabeled target cells (HeLa) with unlabeled extracts, predominantly T antigen from hydroxylamine extracts became 3H labeled by the target cells, dependent on metabolic or enzymatic conditions. 3H-labeled T antigen became unlabeled after treatment with hydroxylamine indicating a covalent ester linkage between cell surface-bound T antigen and lipid of the target cells. The cell surface localization of in vitro acylated T antigen was demonstrated by mild trypsin digestion of living target cells. These results strongly support the idea about a novel mechanism by which a minor subclass of T antigen after being bound to the cell surface becomes covalently linked to lipid of the living cell.
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PMID:Cell surface binding simian virus 40 large T antigen becomes anchored and stably linked to lipid of the target cells. 608 50

Fatty acid synthetases isolated from all mammalian tissues synthesize predominately palmitic acid. However, in vivo the mammary gland fatty acid synthetases of some species are responsible for the synthesis of medium chain fatty acids. The objective of this presentation is to outline the mechanism which regulates the product specificity of fatty acid synthetases in general and to illustrate how this control is modified in the mammary gland. Fatty acid synthetases isolated from mammalian tissues are composed of two similar, probably identical, polypeptides, each carrying as many as seven enzyme components. Thioesterase I, the component which functions to terminate growth of acyl chains on the multienzyme, is located at one terminus of each polyfunctional polypeptide and can be detached by limited proteolysis with trypsin. By studying separately the kinetics of chain elongation by the core of the trypsinized complex and of chain termination by the isolated thioesterase I component, it has been possible to establish that the specificities of the elongation and termination reactions account for the synthesis of predominantly the carbon-16 fatty acid by purified fatty acid synthetases. Mammary glands of some species contain an additonal enzyme, thioesterase II, which can modify the product specificity of fatty acid synthetase by hydrolyzing medium chain acyl moieties from thioester linkage to the 4'phosphopantetheine of the multienzyme. At all stages of development of rat mammary gland, the amount of theoesterase II present correlates well with the proportion of medium chain fatty acids synthesized by the gland. This mammary gland-specific thioesterase appears responsible for the ability of this tissue to synthesize medium chain fatty acids characteristic of milk fat.
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PMID:Mechanism of chain length determination in biosynthesis of milk fatty acids. 610


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